In recent years, with the rapid progress of research concerning the structures and functions of sugar chains, research efforts have been undertaken to develop applications of oligosaccharides, glycolipids, glycoproteins, and similar materials having physiological activities in the fields of drugs and functional materials. Among sugar chains, a sialic-acid-containing sugar chain having N-acetylneuraminic acid (NeuAc) at an end thereof plays an important role as a receptor in, for example, cell adhesion or viral infection.
Generally, the sialic-acid-containing sugar chain is synthesized by use of sialyltransferase as a catalyst. Sialyltransferase is an enzyme which catalyzes the transfer of sialic acid from CMP-N-acetylneuraminic acid (CMP-NeuAc), which serves as a sugar donor, to an acceptor such as a sugar chain.
However, CMP-NeuAc employed as a sugar donor is very expensive and therefore has been provided only in small amounts on reagent levels.
In a known method for producing CMP-NeuAc, CMP-NeuAc is synthesized from cytidine 5′-triphosphate (CTP) and NeuAc serving as substrates by use of CMP-NeuAc synthase as a catalyst (Appl. Microbiol. Biotechnol., 44, 59-67(1995)). Since CTP and NeuAc are expensive substances, direct use of these substances as starting materials inevitably increases the cost for producing CMP-NeuAc.
Recently, Koizumi et al. have developed a process for producing CMP-NeuAc from orotic acid and NeuAc as starting materials by using, in combination, Brevibacterium ammoniagenes cells which transform orotic acid to uridine 5′-triphosphate (UTP), a recombinant E. coli which produces a CTP synthase that catalyzes transformation of UTP to CTP, and a recombinant E. coli which produces a CMP-NeuAc synthase (Appl. Microbiol. Biotechnol., 53, 257-261, (2000)). This process does not employ expensive CTP. However, cumbersome steps and large-scale facilities must be provided for preparing cells of a plurality of species, and NeuAc, which is an expensive reagent, is still employed, discouraging employment of the process in practice.
Meanwhile, regarding the method for producing NeuAc, there has been known a process where colominic acid—a polymer of sialic acid—is recovered from a microorganism, and NeuAc is obtained through chemical decomposition of colominic acid. Recently, some processes employing an enzyme have also been developed.
Examples of such enzymatic processes include
(1) a process for producing NeuAc from N-acetylmannosamine (ManNAc) by use of NeuAc lyase or NeuAc synthase (J. Am. Chem. Soc., 110, 6481 (1988), J. Am. Chem. Soc., 110, 7159 (1988), and Japanese Patent Application Laid-Open (kokai) No. 10-4961);
(2) a process for producing NeuAc through transformation of N-acetylglucosamine (GlcNAc) to N-acetylmannosamine (ManNAc) under alkaline conditions and subsequent treatment of ManNAc with NeuAc lyase or NeuAc synthase (Japanese Patent Application Laid-Open (kokai) No. 5-211884, Biotechnology And Bioengineering, Vol. 66, No. 2 (1999), and Enzyme Microb. Technol., Vol. 20 (1997)); and
(3) a process for producing NeuAc from GlcNAc by use of N-acetylglucosamine (GlcNAc) 2-epimerase which catalyzes transformation of GlcNAc to ManNAc, and NeuAc lyase or NeuAc synthase (WO95/26399, Japanese Patent Application Laid-Open (kokai) Nos. 3-180190, and 2001-136982).
However, these processes have drawbacks. The process (1) employs ManNAc, which is an expensive starting material. Process (2) includes a cumbersome step for isolating ManNAc from a mixture of GlcNAc and ManNAc, although the process employs inexpensive GlcNAc as a starting material. The problem with process (3) resides in that, as shown in the following scheme, it employs GlcNAc 2-epimerase, which functions only in the presence of ATP. Thus, expensive ATP must be used, or ATP must be produced from adenine—a precursor of ATP—by use of a microorganism, making the process unsatisfactory.
<Process (3)>
